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Figure 5.9

The use ofI3C NMR spectroscopy in demonstrating the labelling of the methyl ester of methyl 6-methylsalicylate. (a) The proton-decoupled spectrum of the unlabelled compound, (b) Substitution of deuterium for hydrogen on a methyl group causes UC-2H coupling and a small upfield shift (0.9 Hz) for CD3. (c). The appearance of the ester methyl of the compound after 15% incorporation of deuterium as in Figure 5.8. The coupled CD3 and singlet CH3 almost overlap the conditions for its isolation, and the best conditions for feeding with sodium acetate. Then a mixture of sodium [l-13C]acetate, [2-13C]acetate and unlabelled acetate were fed to the bacterium. The 13C NMR spectrum of the isolated product showed labelling in carbon atoms 1, 2, 6 and 7 only (Figure 5.10 (b)). Therefore, these atoms were derived from intact acetate units. The result suggested an experiment with [l-13C]isovaleric acid, which gave an enriched NMR peak for carbon atom 8 (Figure 5.10 (c)). The experimenters then thought that the perhaps the remaining three atoms came from glycerol, so they synthesized glycerol labelled at carbon atoms 1 and 3. As expected the NMR spectrum then showed enhanced signals at carbon atoms 4 and 5 of VBA (Figure 5.10 (<d)). This time there was some labelling at carbon atoms 2 and 7 because there was

Figure 5.10 (a) The structure and numbering of virginae butanolide A. (b) Labelling introduced by supplying a mixture ofI3CH3COOH, CH313COOH and CH3COOH. (c) Labelling with [1-13C] isovaleric acid, (d) Effect of feeding with [1,3-'3C2]glycerol, (e) Incorporation intact of a ready-made (1-ketoester some degradation of the glycerol to 13CH3COOH, and incorporation of this (Figure 5.10 (,b)). The 13C NMR spectrum does not tell whether atoms 4 and 5 are in the same molecule, an important point for a rigorous proof, but the mass spectrum did show that molecules of VBA were either unlabelled or twice-labelled with 13C. Finally to show that the isovaleric acid and the acetate units were linked into a kind of polyketide before linking to glycerol, a di-labelled thioester (Figure 5.10 (e)) was prepared (to resemble a CoA thioester) with two adjacent 13C atoms.

The 13C NMR spectrum of unlabelled VBA is shown in Figure 5.11 (a), with the same spectrum (on a much smaller sample) after labelling with [1,3-13C2]glycerol (Figure 5.11 (A)). As well as the enhanced intensity of the C-4 and C-5 atoms (about 6.2%), there was unexpected enrichment of